Enhanced potency of individual and bivalent DNA replicon vaccines or conventional DNA vaccines by formulation with aluminum phosphate.

DNA vaccines against botulinum neurotoxin (BoNTs) induce protective humoral immune responses in mouse model, but when compared with conventional vaccines such as toxoid and protein vaccines, DNA vaccines often induce lower antibody level and protective efficacy and are still necessary to increase their potency. In this study we evaluated the potency of aluminum phosphate as an adjuvant of DNA vaccines to enhance antibody responses and protective efficacy against botulinum neurotoxin serotypes A and B in Balb/c mice. The administration of these individual and bivalent plasmid DNA replicon vaccines against botulinum neurotoxin serotypes A and B in the presence of aluminum phosphate improved both antibody responses and protective efficacy. Furthermore, formulation of conventional plasmid DNA vaccines encoding the same Hc domains of botulinum neurotoxin serotypes A and B with aluminum phosphate adjuvant increased both antibody responses and protective efficacy. These results indicate aluminum phosphate is an effective adjuvant for these two types of DNA vaccines (i.e., plasmid DNA replicon vaccines and conventional plasmid DNA vaccines), and the vaccine formulation described here may be an excellent candidate for further vaccine development against botulinum neurotoxins.

Development and preclinical evaluation of a new F(ab')2 antitoxin against botulinum neurotoxin serotype A.

Concern about the malicious applications of botulinum neurotoxin has highlighted the need for a new generation of safe and highly potent antitoxins. In this study, we developed and evaluated the preclinical pharmacology and safety of a new F(ab')2 antitoxin against botulinum neurotoxin serotype A (BoNT/A). As an alternative to formalin-inactivated toxoid, the recombinant Hc domain of botulinum neurotoxin serotype A (rAHc) was used to immunize horses, and the IgGs from the hyperimmune sera were digested to obtain F(ab')2 antitoxin. The protective effect of the new F(ab')2 antitoxin against BoNT/A was determined both in vitro and in vivo. The results showed that the F(ab')2 antitoxin could prevent botulism in mice challenged with BoNT/A and effectively delayed progression of paralysis from botulism in the therapeutic setting. The preclinical safety of the new F(ab')2 antitoxin was also evaluated, and it showed neither harmful effects on vital functions nor adverse effects such as acute toxicity, or immunological reactions in mice and dogs. Thus, our results provide valuable experimental data for this new antitoxin as a potential candidate for treatment of botulism caused by BoNT/A, and our findings support the safety of the new F(ab')2 antitoxin for clinical use. Our study further demonstrates the proof of concept for development of a similar strategy for obtaining potent antitoxin against other BoNT serotypes.

Neutralizing antibodies of botulinum neurotoxin serotype A screened from a fully synthetic human antibody phage display library.

The botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous protein substances known. The neutralizing antibodies against botulinum neurotoxin can effectively prevent and cure the toxicosis. Using purified Hc fragments of botulinum neurotoxin serotype A (BoNT/A-Hc) as antigen, 2 specific neutralizing antibodies mapping different epitopes were selected from a fully synthetic human antibody library. The 2 antibodies can effectively inhibit the binding between BoNT/A-Hc and differentiated PC-12 cells in vitro, and the neutralization was evaluated in vivo. Although no single mAb completely protected mice from toxin, they both could prolong time to death when challenged with 20 LD(50)s (50% lethal doses) of BoNT/A. When used together, the mAbs completely neutralized 1000 LD(50)s/mg Ab, suggesting their high neutralizing potency in vivo. The results would lead to further production of neutralizing antibody drugs against BoNT/A. It also proved that it was a quick method to obtain human therapeutic antibodies by selecting from the fully synthetic human antibody phage display library.